Caspase-3 and caspase-9 activity was measured by using caspase-3 (cat. no. C1115, Beyotime Institute of Biotechnology) and caspase-9 activity assay kits (cat. no. C1158, Beyotime Institute of Biotechnology). At 48 h post-transfection, the PASMCs (1×106) were harvested and lysed at 4°C for 30 min using radioimmuno-precipitation assay (RIPA) buffer (cat. no. P0013K, Beyotime Institute of Biotechnology). Total protein was quantified using a bicinchoninic acid (BCA) assay, followed by incubation with 10 µl Ac-DEVD-pNA and 10 µl Ac-LEHD-pNA at 37°C for 2 h. The absorbance of each well was measured with a micro-plate reader (Bio-Tek Instruments, Inc.) set at 405 nM.
Caspase 9 activity assay kit
Manufactured by Beyotime
Sourced in China
The Caspase 9 Activity Assay Kit is a laboratory equipment product that measures the activity of the enzyme caspase 9. Caspase 9 is a key regulator of apoptosis, a form of programmed cell death. The kit provides a quantitative colorimetric method for detecting caspase 9 activity in cell lysates, tissue extracts, or purified enzyme preparations.
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Lab products found in correlation
Caspase 3 activity assay kit, by Beyotime (12 mentions)
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Cell counting kit 8 cck 8, by Beyotime (2 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Mqx200, by Agilent Technologies (1 mentions)
Dullbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline tablets, by Avantor (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Elx800, by Agilent Technologies (1 mentions)
Total superoxide dismutase assay kit, by Beyotime (1 mentions)
Luciferase based atp assay kit, by Beyotime (1 mentions)
Glutathione reductase assay kit, by Beyotime (1 mentions)
Cellular glutathione peroxidase assay kit, by Beyotime (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Reactive oxygen species assay kit ros, by Beyotime (1 mentions)
Tunel apoptosis assay kit, by Beyotime (1 mentions)
Hepg2 cell line, by American Type Culture Collection (1 mentions)
32 protocols using caspase 9 activity assay kit
1
Caspase-3 and Caspase-9 Activity Assay
2
Caspase-3 and Caspase-9 Activity Assay
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The Caspase-3 and Caspase-9 activity detections were performed by the Beyotime Caspase-3 and Caspase-9 activity assay kits according to the manufacturer’s instructions in order to further investigate the influence mechanism of MSNCA@GODOX-HA on the intrinsic apoptosis pathway. Briefly, the confluent cells (1×106 cells per well in 6-well plates) were incubated with the culture medium containing different CA preparations (MSNCA@GODOX-HA, MSNCA@GODOX, MSNCA and free CA, adjusted to 40μg/mL according to the concentration of free CA) or DOX preparations (MSNCA@GODOX, GODOX and free DOX solutions, adjusted to 5μg/mL according to the concentration of free DOX) for 24h.25 (link) Subsequently, cells were trypsinized and centrifuged at 1000rpm, then lysed with lysis buffer (50μL) and incubated on ice for 15min. The protein content of cells was extracted by the centrifugation of lysates (16,000–20,000rpm) at 4°C for 10min. The substrate reaction buffers containing Caspase-3 or Caspase-9 (2mM) were added separately to the supernatant and incubated for 1h at 37°C (until the significant changes in color occurred). Then the absorbance was immediately measured at 405 nm by a plate reader (BioTek, USA). The protein content was determined by the Bradford method to normalize the activity of Caspase-3 or Caspase-9, and the measurement results were expressed as the percent of control.
3
Caspase Activity Assay Protocol
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Caspase levels were measured with caspase-3 and caspase-9 activity assay kits according to the manufacturer’s instructions (Beyotime). The absorbance was measured on a microplate reader at 405 nm, and the caspase activities were subsequently calculated based on the absorbance.
4
Caspase-3 and Caspase-9 Activity Assay
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The activities of caspase-3 and caspase-9 in the supernatant were detected using the colorimetric caspase-3 and caspase-9 activity assay kits (Beyotime), respectively. Absorbance at 405 nm was measured with a microplate reader (MQX 200, BioTek Instruments).
5
Cytotoxicity Evaluation of Novel Compounds
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GK was provided by Chengdu Must Bio-Technology Co., Ltd (Sichuan, China). Dullbecco's modified eagle's medium (DMEM) and penicillin-streptomytin was acquired from Gibco (Thermo Fisher Scientific, Grand Island, Shanghai, China). Phosphate-buffered saline (PBS) tablets were purchased from Amresco (Solon, OH, USA). 4′6-Diamidino-2-phenylindole was purchased from Sigma (St. Louis, MO, USA). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) was from Biofroxx (Einhausen, Hessen, Germany). Trypsin-ethylenedimainetetraacetic acidwas obtained from Gibco (Burlington, Ontario, Canada). Fetal bovine serum was purchased from Corning (Medford, MA, USA). Cy5.5 NHS ester was purchased from Ruixibio (Xi'an, China). Calcium chloride (CaCl2) was purchased from China Pharmaceutical Group Chemical Reagent Co., Ltd. (Shanghai, China). Caspase-3 and caspase-9 activity assay kits were purchased from Beyotime Biotechnology (Haimen, China).
6
Caspase-3 and Caspase-9 Activity Assay
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Caspase-3 and caspase-9 activities were assessed by the colorimetric method using Caspase-3 and Caspase-9 activity assay kits (Beyotime Institute of Biotechnology), respectively, according to the manufacturer's protocol. The cells were counted and washed in PBS. The samples were collected by centrifugation (600 × g; 5 min; 4°C), suspended in 100 µl lysis buffer (Beyotime Institute of Biotechnology) and kept on ice for 20 min. Following centrifugation (20,000 × g; 10 min; 4°C), the supernatant was collected for analysis. A total of 2 mM Ac-DEVD-pNA or Ac-LEHD-pNA was used to measure Caspase-3 activity or caspase-9 activity, respectively. The absorbance was measured at 405 nm using a microplate reader, and Caspase-3 and caspase-9 activity was calculated based on the relative value of p-nitroanilide content.
7
Caspase-3 and Caspase-9 Activity Assay
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The activity of caspase-3 and caspase-9 was determined using the caspase-3 activity assay kit (Beyotime Biotechnology) and the caspase-9 activity assay kit (Beyotime Biotechnology) respectively according to the manufacturer’s protocol. Briefly, cell lysates were prepared and incubated with Ac-DEVD-pNA (caspase-3 substrate) or Ac-LEHD-pNA (caspase-9 substrate) in reaction buffer for 2‒4 h at 37°C. Caspase activity was measured by cleavage of the corresponding Ac-DEVD-pNA or Ac-LEVD-pNA into p-nitroanilide (pNA). The absorbance was detected at 405 nm with a microplate reader (ELX-800; BioTek Instruments, Winooski, USA).
8
Evaluating Cellular Antioxidant Markers
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The Caspase 9 Activity Assay Kit (Beyotime, China, Cat. No: C1158) was used to measure the activity of caspase-9 according to the manufacturer’s instructions. The concentrations of GSH, SOD and GPX were evaluated using commercial kits (Cellular Glutathione Peroxidase Assay Kit, Beyotime, China, Cat. No: S0056; Glutathione Reductase Assay Kit, Beyotime, China, Cat. No: S0055; Total Superoxide Dismutase Assay Kit, Beyotime, China, Cat. No: S0101, respectively). ATP production was measured using a luciferase-based ATP assay kit (Beyotime Institute of Biotechnology) with a microplate reader [44 (link)].
9
Caspase-9 and Caspase-3 Activity Assays
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The activity of caspase-9 and caspase-3 in cardiomyocytes or myocardium was measured by the Caspase 9 Activity Assay Kit (C1158; Beyotime, Shanghai, China) and the Caspase 3 Activity Assay Kit (C1116; Beyotime, Shanghai, China), respectively. The experimental operations were in accordance with the manual provided by the company.
10
Quantifying Myocardial Caspase-9 Activity
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Myocardial caspase-9 activity was determined using a caspase-9 activity assay kit (c1158; Beyotime Biotech, Shanghai, China). The protocol was similar to that of the caspase-3 activity assay, except for the use of Ac-LEHD-pNA as a substrate. Caspase-9 activity was expressed as unit/h/mg protein.
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